1. Field of the Invention
The present invention relates generally to the field of biochemical pathways. More particularly, it concerns the pathways connecting DNA damage and phosphorylation by tyrosine kinases.
2. Description of the Related Art
Current treatment methods for cancer, including radiation therapy alone, surgery and chemotherapy, are known to have limited effectiveness. Cancer mortality rates will therefore remain high well into the 21st century. The rational development of new cancer treatment methods will depend on an understanding of the biology of the cancer cell at the molecular level.
Certain cancer treatment methods, including radiation therapy, involve damaging the DNA of the cancer cell. The cellular response to DNA damage includes activation of DNA repair, cell cycle arrest and lethality (Hall, 1988). The signaling events responsible for the regulation of these events, however, remain unclear.
Several checkpoints in cell cycle progression control growth in response to diverse positive and negative regulatory signals (Lau and Pardee, 1982). Ionizing radiation, for example, slows growth by inducing delays in G1/S and G2 phases of the cell cycle. The available evidence suggests that G2 arrest in necessary for repair of DNA damage before entry into mitosis (Steinman et al., 1991; Weinert and Hartwell, 1988). Genetic studies in Saccharomyces cerevisiae have demonstrated that the RAD9 protein controls G2 arrest induced by DNA damage (Schiestl et al., 1989; Murray, 1989). Mutants of the rad9 locus are unable to delay entry into mitosis following exposure to genotoxic agents and thereby replicate damaged DNA. Although the mammalian homolog of rad9 remains unidentified, other studies in various eukaryotic cells have demonstrated that entry into mitosis is regulated by a 34 kD serine/threonine protein kinase, designated p34cdc2 (Nurse, 1990; Pines and Hunter, 1989; Russell and Nurse, 1987).
Recent studies have shown that exposure of eukaryotic cells to ionizing radiation is associated with induction of certain early response genes that code for transcription factors. Members of the jun/fos and early growth response (EGR) gene families are activated by ionizing radiation (Sherman et al., 1990; Datta et al., 1992a). Expression and DNA binding of the nuclear factor kB (NF-kB) are also induced in irradiated cells (Brach et al., 1991; Uckun et al., 1992a). Other studies have shown that levels of the tumor suppressor p53 protein increase during X-ray-induced arrest of cells in Gi phase (Kastan et al., 1991; 1992). The activation of these transcription factors presumably represents transduction of early nuclear signals to longer term changes in gene expression that constitute the response to irradiation. Ionizing radiation also induces protein kinase C (PKC) and protein tyrosine kinase activities (Hallahan et al., 1990; Uckun et al., 1993). However, the specific kinases responsible for these activities and their substrates require further study.
Mitomycin C (MMC) is an antitumor antibiotic isolated from Streptomyces caespitosus that covalently binds to DNA (Tomasz et al., 1988). This agent induces both monofunctional and bifunctional DNA lesions (Carrano et al., 1979). Other studies have demonstrated that MMC stimulates the formation of hydroxyl radicals (Dusre et al., 1989). Although the precise mechanism of action of this agent is unclear, MMC-induced cytotoxicity has been attributed to DNA alkylation and the formation of interstrand cross-links (Carrano et al., 1979; Dusre et al., 1989; Tomasz et al., 1988). Treatment of mammalian cells with MMC is associated with inhibition of DNA synthesis and induction of sister-chromatid exchange (Carrano et al., 1979). Previous work has demonstrated that MMC also enhances transcription of HIV-1 and collagenase promoter constructs transfected into HeLa cells (Stein et al., 1989). These studies indicated that AP-1 is involved in MMC-induced activation of the collagenase enhancer. However, little is known about the effects of this agent on other signaling events.
Protein tyrosine phosphorylation contributes to the regulation of cell growth and differentiation. Protein tyrosine kinases can be divided into receptor-type and nonreceptor-type (Src-like) kinases (Cantley et al., 1991; Hanks et al., 1988; Bonni et al., 1993; Larner et al., 1993; Ruff-Jamison et al., 1993). Several protein tyrosine kinases have been purified from the cytosolic fractions of various tissues (Nakamura et al., 1988; Wong and Goldberg, 1984; Zioncheck et al., 1986).
The Src-like kinases, which can associate with receptors at the plasma membrane, induce rapid tyrosine phosphorylation and/or activation of effectors such as phospholipase C-xcex31 (PLCxcex31) (Carter et al., 1991), PLCxcex32 (Hempel et al., 1992), mitogen-activated protein (MAP) kinase (Casillas et al., 1991), GTPase activating protein (GAP) (Gold et al., 1992a) and phosphati-dylinositol 3-kinase (PI3-K) (Gold et al., 1992b). Recent studies have demonstrated an increase in tyrosine phosphorylation following irradiation of B-lymphocyte precursors (Uckun et al., 1993). Studies of p59fyn, p56/p53lyn, p55blk and p56lck activity demonstrated that these Src-family tyrosine kinases were not responsible for radiation-induced tyrosine phosphorylation (Uckun et al., 1992a). These findings suggested that other protein tyrosine kinases, perhaps of the receptor-type, are involved in the response of cells to ionizing radiation.
Varying the environmental conditions following exposure to ionizing radiation or DNA damaging agents can influence the proportion of cells that survive a given dose due to the expression or repair of potentially lethal damage (PLD). The damage is potentially lethal because while under normal circumstances it causes cell death, manipulation of the post-irradiation environment can modify the cell response. Studies show that cell survival can be increased if the cells are arrested in the cell cycle for a protracted period of time following radiation exposure, allowing repair of DNA damage. (Hall, 1988). Thus, PLD is repaired and the fraction of cells surviving a given dose of x-rays is increased if conditions are suboptimal for growth, such that cells do not have to undergo mitosis while their chromosomes are damaged.
For some diseases, e.g., cancer, ionizing radiation is useful as a therapy. Methods to enhance the effects of radiation, thereby reducing the necessary dose, would greatly benefit cancer patients. Therefore, methods and compositions were sought to enhance radiation effects by increasing the sensitivity of cells to damage from ionizing radiation and DNA damaging agents such as alkylating compounds. Cells that are irradiated or treated with DNA damaging agents halt in the cell cycle at G2, so that an inventory of chromosome damage can be taken and repair initiated and completed before mitosis is initiated. By blocking the stress or survival response in these cells, they undergo mitosis with damaged DNA, express the mutations, and are at a greater risk of dying.
In one aspect, the present invention provides a process of selectively activating the lyn family of Src-like tyrosine kinases in a cell that expresses the lyn gene. In accordance with that process, the cell is exposed to an effective activating dose of ionizing radiation. In a preferred embodiment, p56/p53lyn is activated.
In another aspect, the present invention provides a process of stimulating tyrosine phosphorylation of Src-like tyrosine kinase substrates. In such a process, a cell that expresses the lyn gene is exposed to an effective activating dose of ionizing radiation. Exemplary Src-like tyrosine kinase substrates include phospholipase C-xcex31, phospholipase C-xcex32, mitogen-activated protein kinase, GTPase activating protein, phosphatidylinositol 3-kinase and enolase.
In another aspect, the present invention provides a process of inhibiting ionizing radiation induced tyrosine phosphorylation in a cell that expresses the lyn gene comprising inhibiting the activity of p56/p53lyn. In a preferred embodiment, p56/p53lyn activity is inhibited by exposing the cell to an effective inhibitory amount of herbimycin A or genistein.
The present invention also provides a process of stimulating the activity of p56/p53lyn with out the concomitant stimulation of other Src-like tyrosine kinases by treating cells with genotoxic alkylating agents. In such a process, a cell that expresses the lyn gene is exposed to an effective activating dose of an alkylating agent, for example, mitomycin C. Subsequent to this exposure, the cells are treated with a tyrosine kinase inhibitor that inhibits the activity of p56/p53lyn, preventing phosphorylation on p34cdc2 on tyrosine, effectively allowing the cells to progress past G2 arrest.
The G2 phase is the point in the cell cycle used DNA repair following damage from ionizing radiation or alkylating agents. Other DNA damaging agents also are able to cause cell arrest in the G2 phase. By preventing delays in G2 cells will enter mitosis before the DNA is repaired and therefore the daughter cells will likely die. By lengthening the G2 period, cells undergo repair and survival following exposure to a DNA damaging agent increases.
DNA damaging agents or factors are defined herein as any chemical compound or treatment method that induces DNA damage when applied to a cell. Such agents and factors include ionizing radiation and waves that induce DNA damage, such as, xcex3-irradiation, X-rays, UV-irradiation, microwaves, electronic emissions, and the like. A variety of chemical compounds, also described as xe2x80x9cchemotherapeutic agentsxe2x80x9d, function to induce DNA damage, all of which are intended to be of use in the combined treatment methods disclosed herein. Chemotherapeutic agents contemplated to be of use, include, e.g., alkylating agents such as mitomycin C, adozelesin, cis-platinum, and nitrogen mustard. The invention also encompasses the use of a combination of one or more DNA damaging agents, whether ionizing radiation-based or actual compounds, with one or more tyrosine kinase inhibitors.
To kill a cell in accordance with the present invention, one would generally contact the cell with a DNA damaging agent and a tyrosine kinase inhibitor in a combined amount effective to kill the cell. The term xe2x80x9cin a combined amount effective to kill the cellxe2x80x9d means that the amount of the DNA damaging agent and inhibitor are sufficient so that, when combined within the cell, cell death is induced. Although not required in all embodiments, the combined effective amount of the two agents will preferably be an amount that induces more cell death than the use of either element alone, and even one that induces synergistic cell death in comparison to the effects observed using either agent alone. A number of in vitro parameters may be used to determine the effect produced by the compositions and methods of the present invention. These parameters include, for example, the observation of net cell numbers before and after exposure to the compositions described herein.
Similarly, a xe2x80x9ctherapeutically effective amountxe2x80x9d is an amount of a DNA damaging agent and tyrosine kinase inhibitor that, when administered to an animal in combination, is effective to kill cells within the animal. This is particularly evidenced by the killing of cancer cells within an animal or human subject that has a tumor. xe2x80x9cTherapeutically effective combinationsxe2x80x9d are thus generally combined amounts of DNA damaging agents and tyrosine kinase inhibitors that function to kill more cells than either element alone and that reduce the tumor burden.
The present invention generally provides novel strategies for the improvement of chemotherapeutic intervention. It is proposed that the combination of a DNA damaging agent and a tyrosine kinase inhibitor will lead to synergistic cancer cell killing effects over and above the actions of the individual DNA damaging component.
In certain embodiments, a process of enhancing cell death is provided, which comprises the steps of first treating cells or tumor tissue with a DNA damaging agent, such as ionizing radiation or an alkylating agent, followed by contacting the cells or tumors with a protein kinase inhibitor, preferably a tyrosine kinase inhibitor. Examples of alkylating agents are mitomycin C, adozelesin, nitrogen mustard, cis-platinum. Exemplary tyrosine kinase inhibitors are genistein or herbimycin.
DNA damaging agents or factors are defined herein as any chemical compound or treatment method that induces DNA damage when applied to a cell. Such agents and factors include radiation and waves that induce DNA damage, such as, xcex3-irradiation, X-rays, UV-irradiation, microwaves, electronic emissions, and the like. A variety of chemical compounds, which may be described as xe2x80x9cchemotherapeutic agentsxe2x80x9d, also function to induce DNA damage, all of which are intended to be of use in the combined treatment methods disclosed herein. Chemotherapeutic agents contemplated to be of use, include, e.g., mitomycin C (MMC), adozelesin, cis-platinum, nitrogen mustard, 5-fluorouracil (5FU), etoposide (VP-16), camptothecin, actinomycin-D, cisplatin (CDDP).
The invention provides, in certain embodiments, methods and compositions for killing a cell or cells, such as a malignant cell or cells, by contacting or exposing a cell or population of cells to one or more DNA damaging agents and one or more tyrosine kinase inhibitors in a combined amount effective to kill the cell(s). Cells that may be killed using the invention include, e.g., undesirable but benign cells, such as benign prostate hyperplasia cells or over-active thyroid cells; cells relating to autoimmune diseases, such as B cells that produce antibodies involved in arthritis, lupus, myasthenia gravis, squamous metaplasia, dysplasia and the like. Although generally applicable to killing all undesirable cells, the invention has a particular utility in killing malignant cells. xe2x80x9cMalignant cellsxe2x80x9d are defined as cells that have lost the ability to control the cell division cycle, as leads to a xe2x80x9ctransformedxe2x80x9d or xe2x80x9ccancerousxe2x80x9d phenotype.
To kill cells, such as malignant cells, using the methods and compositions of the present invention, one would generally contact a xe2x80x9ctargetxe2x80x9d cell with at least one DNA damaging agent and at least one tyrosine kinase inhibitor in a combined amount effective to kill the cell. This process may involve contacting the cells with the DNA damaging agent(s) or factor(s) and the tyrosine kinase inhibitor at the same time. This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes the DNA damaging agent and the other composition includes the tyrosine kinase inhibitor.
Naturally, it is also envisioned that the target cell may be first exposed to the DNA damaging agent(s) and then contacted with a tyrosine kinase inhibitor, or vice versa. In such embodiments, one would generally ensure that sufficient time elapses, so that the two agents would still be able to exert an advantageously combined effect on the cell. In such instances, it is contemplated that one would contact the cell with both agents within about 12 hours of each other, and more preferably within about 6 hours of each other, with a delay time of only about 4 hours being most preferred. These times are readily ascertained by the skilled artisan.
The terms xe2x80x9ccontactedxe2x80x9d and xe2x80x9cexposedxe2x80x9d, when applied to a cell, are used herein to describe the process by which a tyrosine kinase inhibitor, such as genistein or herbimycin A, and a DNA damaging agent or factor are delivered to a target cell or are placed in direct juxtaposition with the target cell. To achieve cell killing, both agents are delivered to a cell in a combined amount effective to kill the cell, i.e., to induce programmed cell death or apoptosis. The terms, xe2x80x9ckillingxe2x80x9d, xe2x80x9cprogrammed cell deathxe2x80x9d and xe2x80x9capoptosisxe2x80x9d are used interchangeably in the present text to describe a series of intracellular events that lead to target cell death.
Tyrosine kinases participate in diverse signalling pathways, and also participate in signal transduction in smooth muscle. xe2x80x9cGenisteinxe2x80x9d, as used herein, refers to the compound described in the Merck Index (7th Edition, 1960, p 474), or a derivative or analogue thereof that functions as a tyrosine kinase inhibitor. The ability of a genistein analogue to inhibit a tyrosine kinase, may be readily determined by methods known to those of skill in the art, as described, for example in Akiyama et al. (1987), incorporated herein by reference. Also encompassed are genistein-like compounds that form an active tyrosine kinase inhibitor upon ingestion.
The present invention also provides advantageous methods for treating cancer that, generally, comprise administering to an animal or human patient with cancer a therapeutically effective combination of a DNA damaging agent and a tyrosine kinase inhibitor. Chemical DNA damaging agents and/or inhibitors may be administered to the animal, often in close contact to the tumor, in the form of a pharmaceutically acceptable composition. Direct intralesional injection is contemplated, as are other parenteral routes of administration, such as intravenous, percutaneous, endoscopic, or subcutaneous injection.
In terms of contact with a DNA damaging agent, this may be achieved by irradiating the localized tumor site with ionizing radiation such as X-rays, UV-light, xcex3-rays or even microwaves. Alternatively, the tumor cells may be contacted with the DNA damaging agent by administering to the animal a therapeutically effective amount of a pharmaceutical composition comprising a DNA damaging compound, such as mitomycin C, adozelesin, cis-platinum, and nitrogen mustard. A chemical DNA damaging agent may be prepared and used as a combined therapeutic composition, or kit, by combining it with a tyrosine kinase inhibitor, as described above.
Other embodiments of the invention concern compositions, including pharmaceutical formulations, comprising a DNA damaging agent in combination with a tyrosine kinase inhibitor. These compositions may be formulated for in vivo administration by dispersion in a pharmacologically acceptable solution or buffer. Suitable pharmacologically acceptable solutions include neutral saline solutions buffered with phosphate, lactate, Tris, and the like. Preferred pharmaceutical compositions of the invention are those that include, within a pharmacologically acceptable solution or buffer, mitomycin C in combination with genistein or herbimycin A.
Still further embodiments of the present invention are kits for use in killing cells, such as malignant cells, as may be formulated into therapeutic kits for use in cancer treatment. The kits of the invention will generally comprise, in suitable container means, a pharmaceutical formulation of a DNA damaging agent and a pharmaceutical formulation of a tyrosine kinase inhibitor. These agents may be present within a single container, or these components may be provided in distinct or separate container means.
The components of the kit are preferably provided as a liquid solution, or as a dried powder. When the components are provided in a liquid solution, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred. When reagents or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.
Although kits have been described as part of this invention, it should be noted that the use of ionizing radiation to create DNA damage is an important aspect of the invention not specifically provided in kit form.